We also examined the enhancing activity of sesquiterpenoids with antibiotics against E. coli (ATCC 25922), which as a gram-negative bacterium possesses the additional permeability barrier of an outer membrane (OM) structure. The effect of nerolidol or farnesol on the antimicrobial activity of polymyxin B against E. coli was determined at various intervals by pour plating. Cells were grown and diluted in phosphate buffer as described above for S. aureus and treated with a 1 mM concentration of either sesquiterpenoid and/or 10 μg of polymyxin B sulfate (Sigma; 8,100 units mg−1) ml−1. Cell suspensions were incubated in a 37°C water bath for up to 30 min, and samples were removed at appropriate intervals, diluted in the same buffer, and enumerated by pour plating with Trypticase soy agar tempered to 50°C. Plates were incubated for 24 h at 37°C prior to counting of colonies. Cells of E. coli treated with sesquiterpenoid and polymyxin B were inactivated more rapidly than were cells treated with polymyxin alone. Figure illustrates this enhancing activity for nerolidol. Under the conditions of this assay, sesquiterpenoids alone had no effect on the viability of this organism.
FIG. 3. Synergistic effect of nerolidol and polymyxin B against E. coli incubated at 37°C in neutral phosphate buffer. Symbols: ○, 1 mM nerolidol; •, 10 μg of polymyxin B ml−1; □, 1 mM nerolidol plus 10 μg (more ...)
Due to their inherent lipophilicities, terpenoid compounds show an affinity for and partition within biological membranes, where their accumulation may impact substantially on the structural and functional properties of these membranes (15
). Our results indicate that the sesquiterpenoid compounds nerolidol, farnesol, bisabolol, and apritone can disrupt the normal barrier function of the bacterial cell membrane, allowing the permeation into the cell of exogenous solutes such as ethidium bromide and antibiotics. This effect is more pronounced for gram-positive bacteria, probably due to the lack of additional permeability barriers, particularly the OM of gram-negative bacteria. Still, the polymyxin results observed in this study suggest that sesquiterpenoids may also find applications in sensitizing gram-negative bacteria to other antibiotics or antimicrobials, provided that steps are taken to enhance the permeability of the OM or that antibacterial agents already possessing some OM-permeabilizing activities are used. Thus, it would be of interest to evaluate the sensitizing activities of these sesquiterpenoids in combination with compounds known to disrupt the OM (16
In preliminary work, we also screened a number of cyclic and acyclic monoterpenoids for their abilities to enhance the susceptibility of S. aureus
to the same antibiotics tested with the sesquiterpenoids. However, these compounds demonstrated little, if any, effect as enhancing agents (data not shown). The increased effectiveness of sesquiterpenoids as enhancers of membrane permeability may stem from their structural resemblance to membrane lipids (e.g., linear molecules with internal lipophilic character and a more polar terminus). Cornwell and Barry (2
) found nerolidol and farnesol to be more effective as skin penetration enhancers than bisabolol and proposed that the longer hydrocarbon tails of these molecules play an important functional role in promoting greater interaction with the interior of the bilayer than that promoted by other classes of terpenoids.
The antibiotics used against S. aureus in this study were selected on the basis of their clinical importance. Other than having primary activities against cytoplasmic targets, they have no unifying themes of structure or specific action. The antibiotics range in molecular weight from 331.3 (ciprofloxacin) to 1,449.2 (vancomycin), and they vary in character from lipophilic (erythromycin) to hydrophilic (gentamicin). The ability of sesquiterpenoid treatment to sensitize bacterial cells to such a heterogeneous group of antibiotics underlines the nonspecific and potentially general nature of this enhancing activity.
We expect that other classes of cytoplasmically targeted antimicrobial agents including food preservatives, biocides, and sanitizers as well as other antibiotics will also show enhanced antimicrobial activities in the presence of these sesquiterpenoids. However, because the principal interactions of terpenoids are with the cytoplasmic membrane, it is likely that the effects of membrane-active antimicrobial compounds will be especially enhanced. The results reported here for gentamicin and polymyxin B support this theory. In addition to its interaction with bacterial ribosomes, the cationic aminoglycoside gentamicin has also been shown to have membrane-destabilizing properties (10
), and polymyxin B is well known for its actions against both the outer and cytoplasmic membranes (4
). Additional work in this laboratory has shown that 2 mM nerolidol dramatically enhances the susceptibility of Listeria monocytogenes
to a variety of membrane-targeted antimicrobials, including monolaurin, hop β-acids, and sodium desoxycholate (B. F. Brehm-Stecher and E. A. Johnson, unpublished results).
Our results suggest a general role for the use of sesquiterpenoid compounds as enhancers of nonspecific bacterial permeability to antibiotics and antimicrobials. In this capacity, these compounds may be useful to the medical, food, and sanitation industries, especially in topical or surface applications, where the required concentrations of sesquiterpenoids could reasonably be achieved. Potential applications include the treatment or prevention of infections, enhancement of food safety, and the fortification of existing sanitizing procedures. Since nerolidol, farnesol, and certain other sesquiterpenoids are generally recognized as safe (GRAS), these compounds may have special applications in concert with antimicrobials intended for use in foods or on food contact surfaces.